Acetic Acid Bacteria

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Food Control 22 (2011) 647e656

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Food Control

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Review

Importance of acetic acid bacteria in food industry

Ilkin Yucel Sengun a,*, Seniz Karabiyikli b,1

a Ege University, Engineering Faculty, Food Engineering Department, 35100 Bornova, Izmir, TurkeybGaziosmanpasa University, Faculty of Engineering and Natural Science, Department of Food Engineering, 60250 Tokat, Turkey

a r t i c l e i n f o

Article history:Received 27 May 2010Received in revised form27 October 2010Accepted 6 November 2010

Keywords:Acetic acid bacteriaIsolationIdentificationVinegarWine

* Corresponding author. Tel.: þ90 232 3113028; faxE-mail addresses: [email protected], ilkin.sen

[email protected], [email protected] Tel.: þ90 3562521616/3285.

0956-7135/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.foodcont.2010.11.008

a b s t r a c t

Acetic acid bacteria (AAB) comprise a group of gram-negative or gram-variable, ellipsoidal to rod-shapedcells that have an obligate aerobic metabolism with oxygen as the terminal electron acceptor. In the firstclassification of AAB, two main genera were determined as Acetobacter and Gluconobacter, but nowadaystwelve genera are recognized and accommodated to the family Acetobacteraceae, the Alphaproteobac-teria: Acetobacter, Gluconobacter, Acidomonas, Gluconacetobacter, Asaia, Kozakia, Swaminathania, Saccha-ribacter, Neoasaia, Granulibacter, Tanticharoenia and Ameyamaea. Isolation, purification, identification andpreservation of AAB are very difficult. Phenotypic methods based on physiological abilitiesies have beenused for identification of AAB by using various media. These phenotypic properties have now beencomplemented or replaced by molecular techniques, which are DNA and RNA based techniques.

AAB are widespread in nature on various plants (fruits, cereals, herbs, etc.). They are importantmicroorganisms in food industry because of their ability to oxidize many types of sugars and alcohols toorganic acids as end products during fermentation process. The best known industrial application of AABis vinegar production. This group of bacteria is also used in cellulose and sorbose production. On theother hand, the oxidizing ability of AAB could have spoilage effect in some products such as in wine. Theaim of the present review is to introduce the importance of AAB in food industry by showing theircurrent taxonomy, enumeration, isolation and identification methods, isolation sources and beneficialeffects in food production systems.

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1. Introduction

Acetic acid bacteria (AAB) are gram-negative or gram-variable,aerobic, non-spore forming, ellipsoidal to rod-shaped cells that canoccur single, in pairs or chains. Their sizes vary between 0.4e1 mmwide and 0.8e4.5 mm long. They are catalase positive and oxidasenegative. The optimumpH for the growth of AAB is 5e6.5while theycan grow at lower pH values between 3 and 4 (Holt, Krieg, Sneath,Staley, & Williams, 1994). AAB are heterogeneous assemble,comprising both peritrichously and polarly flagellated organisms(Gonzales, 2005).

In the early years, AAB were classified into two main genera:Acetobacter and Gluconobacter, but nowadays twelve genera arerecognized and accommodated to the family Acetobacteraceae, theAlphaproteobacteria: Acetobacter, Gluconobacter, Acidomonas, Glu-conacetobacter, Asaia, Kozakia, Swaminathania, Saccharibacter, Neo-asaia, Granulibacter, Tanticharoenia and Ameyamaea. Occurrence of

: þ90 232 [email protected] (I.Y. Sengun),du.tr (S. Karabiyikli).

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Acidomonas, Kozakia, Swaminathania, Saccharibacter, Neoasaia,Granulibacter, Tanticharoenia andAmeyamaea strains is rather rare incommon isolation sources such as vinegar, wine, fruits and flowers(Yamada & Yukphan, 2008; Yukphan et al., 2009, 2008).

AAB have traditionally been enumerated by quantifying viablecolonies by plating in solid culturemedia. Not all themedia supportgrowth of AAB equally and they are selective for one strain oranother (Gullo, Caggia, De Vero, & Giudici, 2006). Thus, differentmedia were used for isolation and phenotypic methods based onphysiological abilities were used for identification. On the otherhand, there are some limitations for plating such as time require-ment, inability to detect viable but noncultivable (VBNC) cells. Toovercome these disadvantages of culturing, new techniques havebeen developed using molecular approaches (Gonzalez, Guillamon,Mas, & Poblet, 2006). AAB were studied genotypically byDNAerRNA hybridization, DNAeDNA hybridization and ribosomalRNA gene sequences (5S rRNA, 16S rRNA, and 23S rRNA).

AAB are well known for the ability to oxidize the sugars andalcohols, resulting an accumulation of organic acids as final prod-ucts. A considerable number of AAB can oxidize alcohols intosugars; mannitol into fructose; sorbitol into sorbose or erythritolinto erythrulose (Gonzales, 2005). Gluconobacter is an industrially

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important genus for the production of L-sorbose from D-sorbitol; D-gluconic acid, 5-keto- and 2-keto-D-gluconate from D-glucose; anddihydroxyacetone from glycerol (Gupta, Singh, Qazi, & Kumar,2001). AAB are involved in some important industrial process (DeVero, 2010; Raspor & Goranovic, 2008). These bacteria canproduce high concentrations of acetic acid from ethanol, whichmakes them important to the vinegar industry. The other knownapplication of AAB is to produce sorbose and cellulose (Gonzales,2005). On the other hand, AAB are sometimes involved in foodsand beverages in detrimental way, such as in wine (Bartowsky &Henschke, 2008). The present review is focused on the taxonomy,isolation and identification of AAB and occurrence or usage of thisgroup in foods and beverages.

2. Taxonomy of AAB

The taxonomy of AAB has not been fully established yet andrearrangements of the group are still in progress. The reasons forthis taxonomic uncertainty are both due to the limited knowledgeof the AAB phylogenesis and isolation, identification and preser-vation difficulties of these bacterial strains (De Vero & Giudici,2008).

The taxonomy of AAB initially based on morphological andphysiological criteria. The first classifications were proposed byHansen in 1894, based on the occurrence of a film in the liquidmedia, and its reaction with iodine. Asai (1934e1935) formulatedthe proposal of classifying AAB into two genera: Acetobacter andGluconobacter. A major change in the classification of AAB wasintroduced by Yamada, Hoshino, and Ishikawa (1997, 1998), inwhich they transferred Acetobacter species containing Q-10 (Ace-tobacter xylinus, Acetobacter liquefaciens, Acetobacter hansenii,

Table 1Species of the twelve genera of acetic acid bacteria.

Genus Species Genus Species

Acetobacter A. acetiA. cerevisiaeA. cibinongensisA. estunensisA. indonesiensisA. lovaniensisA. malorumA. nitrogenifigensA. oeniA. orientalisA. orleanensisA. pasteurianusA. peroxydansA. pomorumA. syzygiiA. tropicalis

Gluconacetobacter Ga. azotocaptansGa. diazotrophicusGa. entaniiGa. europaeusGa. hanseniiGa. intermediusGa. johannaeGa. liquefaciensGa. nataicolaGa. oboediensGa. rhaeticusGa. sacchariGa. saccharivoransGa. swingsiiGa. xylinus

Acidomonas Ac. methanolica Granulibacter Gr. bethesdensisAsaia As. bogorensis

As. krungthrpensisAs. lannensisAs. siamensisAs. spathodeae

Gluconobacter G. albidusG. cerinusG. frateuriiG. japonicusG. kondoniiG. oxydansG. roseusG. sphaericusG. thailandicusG. wancherniae

Ameyamaea Am. chiangmaiensis Saccharibacter S. floricolaNeoasaia N. chiangmaiensis Swaminathania Sw. salitoleransKozakia K. baliensis Tanticharoenia T. sakaeratensis

Table is adapted from Yamada and Yukphan (2008) with combining data fromMalimas et al. (2007) (for G. kondonii); Malimas et al. (2008) (As. lannensis); Malimaset al. (2008a) (for G. roseus); Malimas et al. (2008b) (for G. sphaericus); Yukphan et al.(2008) (for Tanticharoenia); Malimas et al. (2009) (for G. japonicus); Yukphan et al.(2009) (for Ameyamaea); Kommanee et al. (2010) (for As. spathodeae); Yukphanet al. (2010) (for G. wancherniae).

Acetobacter diazotrophicus and Acetobacter europaeus) to the genusGluconacetobacter. Nowadays, the family Acetobacteraceae accom-modates twelve genera for the AAB: Acetobacter, Gluconobacter,Acidomonas, Gluconacetobacter, Asaia, Kozakia, Swaminathania,Saccharibacter, Neoasaia, Granulibacter, Tanticharoenia and Ameya-maea (Yamada & Yukphan, 2008; Yukphan et al., 2009, 2008).Table 1 shows the current classification of AAB.

In general, AAB belonging to the genus Acetobacter have beenfound more frequently than those belonging to Gluconobacter(Camu et al., 2007). Themain differences between these two generawere both cytological and physiological. The main physiologicaldifference was that Acetobacter oxidized ethanol into acetic acidand, subsequently, completed the oxidation of acetic acid intowater and CO2. On the other hand, Gluconobacter species wereunable to complete this oxidation of acetic acid (Gonzales, 2005).Other differential characteristics of AAB are shown in Table 2.

Although the tests given in Table 2 are used to differentiate AABgenera, these phenotypic tests alone are not really reliable and notrecommended (Cleenwerck & de Vos, 2008). As it can be seen fromthe table, AAB are heterogeneous assemble, comprising bothperitrichously and polarly flagellated organisms. In the early years,flagellation pattern was used in the classification of AAB. Thepolarly flagellated AAB species are classified in the genus Gluco-nobacter, while the peritrichously flagellated species are groupedinto the genus Acetobacter (Leifson, 1954). Quinone analyses canalso be used to differentiate AAB genera, as a chemotaxonomicalmethod. The genus Acetobacter is characterized chemotaxonomi-cally by Q-9 as a major respiratory quinone, which is quite uniqueand exceptional, although other genera have Q-10 (Yamada, Aida, &Uemura, 1969). On the other hand, genus-level identification ofAAB using phenotypic tests is relatively easy when compared withspecies level identification (Cleenwerck & de Vos, 2008).

AAB have two enzymes, which play a role in oxidation process:alcohol dehydrogenase and aldehyde dehydrogenase. The alcoholdehydrogenase activity of Acetobacter is more stable under aceticconditions than that of Gluconobacter, which explains why Aceto-bacter produces more acetic acid (Matsushita, Toyama, & Adachi,1994). Acetic acid resistance of AAB is strain dependent (Nanba,Tamura, & Nagai, 1984). Trcek, Raspor, and Teuber (2000) repor-ted that the resistance to acidic environment (pH 2.5e3.5) and therequirements of acetic acid for growth are variable phenotypictraits in A. europaeus strains.

AAB can metabolize a variety of organic acids, such as acetic,citric, fumaric, lactic, malic, pyruvic and succinic acids and thisability brings them important in winemaking (Gonzales, 2005).Different carbohydrates can be used by AAB as carbon sources.Sugar is more preferred as a carbon source by Gluconobacter thanAcetobacter because the species of Gluconobacter can obtain energymore efficiently by the metabolisation of the sugars via pentosephosphate pathway. They oxidize glucose via two alternate path-ways. One operates inside the cell followed by oxidation via thepentose phosphate pathway and second operates outside the celland involves the formation of gluconic acid and ketogluconic acid(Kulka &Walker, 1954; Olijve & Kok, 1979). The former is carried byNADPþ-dependent glucose dehydrogenase, and the latter is per-formed by NADPþ independent glucose dehydrogenase and is alsocalled as “direct glucose oxidation” pathway (Kitos, Wang, Mohler,King, & Cheldelin, 1958). Direct oxidation metabolism pathwayworks only in the presence of >15 mM glucose in the culturemedium (Weenk, Olijve, & Harder, 1984). Few species are able togrow at elevated sugar concentration e.g. Gluconacetobacter diazo-trophicus species can grow at 30% of D-glucose (Swings, 1992).Glucose tolerance of AAB strains isolated from Traditional BalsamicVinegar was studied and the results showed that majority of theisolated strains are inhibited by 25% of glucose while ethanol

Table 2Differential characteristics of the twelve genera of acetic acid bacteria.

Characteristic A Ac As Ga G K S Sa N Gr T Am

Flagellation pe/n n pe/n pe/n po/n n pe n n n n poOxidation of ethanol to acetic acid þ þ �/wa þ þ þ þ v þ v þ þOxidation of acetic acid to CO2 and H2O þ þ þ þb � w w � � w � þOxidation of lactate to CO2 and H2O þ w þ þ/� � w w w � þ � wGrowth on 0.35% acetic acid-containing medium þ þ � þ þ þ þ � þ Nd þ þGrowth on methanol �/wc þ � � � � � � � þ � wGrowth on D-mannitol þ/� w þ/� þ/� þ þGrowth in the presence of 30% D-glucose � � þ þ/� �/þ � Nd þ þ Nd þ �Production of cellulose � � � þ/� � � Nd � Nd NdProduction of levan-like mucous substance from sucrose �/þ � � �/þ � þ Nd � � Nd � �Fixation of molecular nitrgen � � � �/þ � �Ketogenesis (dihydroxyacetone) from glycerol þ/� w �/w þ/� þ þ þ � w � þ wAcid production from:D-Mannitol �/þ � þ/� þ/� þ � � þ w � � �Glycerol �/þ � þ þ þ þ þ � þ v þ wRaffinose � Nd � � þ Nd � þ NdCellular fatty acid type C18:1 C18:1 C18:1 C18:1 C18:1 C18:1

Ubiquinone type Q-9 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10DNA base composition (mol% Gþ C) 52e60 63e66 59e61 55e66 55e63 56e57 57e60 52e53 63.1 59 66.0 65.6

Abbreviations: A, Acetobacter; Ac, Acidomonas; As, Asaia; Ga, Gluconacetobacter; G, Gluconobacter; K, Kozakia; S, Swaminathania; Sa, Saccharibacter; N, Neoasaia; Gr,Granulibacter; T, Tanticharoenia; Am, Ameyamaea; pe, peritrichous; po, polar; n, none; þ, 90% or more of the strains positive; w, weakly positive reaction; �, 90% or more ofthe strains negative; v, variable; Nd, not determined.

a Asaia does not produce acetic acid from ethanol with the exception of one strain producing acid weakly (Yamada et al., 2000).b Overoxidation of acetate to CO2 and H2O depends on acetate concentration in medium.c A. pomorum assimilates methanol weakly (Sokollek et al., 1998b).

Table is adapted from Sievers and Swings (2005) with combining data from Loganathan and Nair (2004) (for Swaminathania); Jojima et al. (2004) (for Saccharibacter); Yukphanet al. (2005) (for Neoasaia); Greenberg et al. (2006) (for Granulibacter) Yukphan et al. (2008) (for Tanticharoenia); Yukphan et al. (2009) (for Ameyamaea).

I.Y. Sengun, S. Karabiyikli / Food Control 22 (2011) 647e656 649

concentration of the cooked and fermented must is less significantfor AAB growth (Gullo et al., 2006).

AAB are mesophilic microorganisms and their optimum growthtemperature is between 25 and 30 �C (Gullo & Giudici, 2008).Thermoresistant properties of AAB isolated from tropical productsof Sub-Saharan Africa were studied by Ndoye et al. (2006). In thestudy, two specific AAB, namely Acetobacter tropicalis and Aceto-bacter pasteurianus were selected for their ability of growth andacetate production at higher temperature. The advantages of thesestrains were reported as the considerable reduction of the coolingwater expenses and the availability of the strains for traditionalvinegar fermentation (Ndoye et al., 2006).

Some AAB species can fix atmospheric nitrogen. Ga. diazo-trophicus, Gluconacetobacter azotocaptans and Ga. johannae areknown as nitrogen-fixing species. In recent years, S. salitolerans,A. peroxydans and A. nitrogenifigenswere also identified as nitrogen-fixing bacteria (Dutta & Gachhui, 2006; Loganathan & Nair, 2004;Muthukumarasamy et al., 2005).

Table 3Different media used for isolation and/or identification of acetic acid bacteria.

Media Proportion

YPMa Medium (Gullo & Giudici, 2008)Yeast extract 0.5%Peptone 0.3%Mannitol 2.5%Agar 1.2%GYCb Medium (Gullo & Giudici, 2008)Glucose 10.0%Yeast extract 1.0%CaCO3 2.0%Agar 1.5%GYc Medium (Yamada & Yukphan, 2008)Glucose 2 gYeast extract 1 gAgar 2 gDistilled water 100 ml

a Yeast extract peptone mannitol medium.b Glucose yeast extract CaCO3 medium.c Glucose yeast extract medium.

3. Enumeration, isolation and identification of AAB

Enumeration, isolation, identification and preservation of AABare not easy. It is important how to isolate pure defined strains fromvarious sources of potential habitats. Not all the media supportgrowthofAABequallyand theyare selective forone strain toanother(Gullo et al., 2006). Although there are lots of media, developed forisolation and/or identification of AAB (Table 3), they mainly consistof the same ingredients with varying proportions, which causedifferent reactions on the plate. By the way, mainly used incubationcondition for the growth of AAB is 30 �C for 2e5 days(Seearunruangchai et al., 2004; Yamada & Yukphan, 2008). AABstrains that are able to grow on solid media may have greatermetabolic fitness. The halo formation that is one of the basic char-acteristics that associates a given colony to the AAB group(Cleenwerck & de Vos, 2008). Glucose Yeast Extract CaCO3 Medium(GYC) was proposed as a medium that enabled most strains to berecovered in traditional vinegars (Gullo et al., 2006). Environment of

Media Proportion

AE-medium (Yamada et al., 1999)Glucose 0.5%Yeast Extract 0.3%Peptone 0.4%Agar 0.9%Absolute ethanol 3 mlAcetic acid 3 mlReinforced AE-medium (Zahoor et al., 2006)Glucose 4%Yeast extract 1%Peptone 1%Na2HPO4$H2O 0.338%Citric acid 0.15%Ethanol 2%(v/v)Acetic acid 1%(v/v)


the isolates is also important for selecting the isolation of suitablemedia. It is reported that isolates from cider or wine vinegarfermentations grew readily in Reinforced AE-Medium (RAE-Medium)while AE-Mediumprovedmost suitable for the cultivationof strains isolated from spirit vinegar fermentations (Sokollek et al.,1998a). In a liquid medium such as wine with high alcohol content,the presence of free SO2 and the low availability of oxygen subjectthe microorganisms to serious stress and they probably need somerecovery before they can grow on a solid medium with a differentcarbon source. In fact, oxygen enrichment has been proposed asa way of improving cultivability, or of putting an end to the VBNCstatus (Millet & Lonvaud-Funel, 2000).

Rapid method for total, viable and non-viable AAB determina-tion was developed by Baena-Ruano et al. (2006) as a possibleoption, using the direct counting in a Neubauer chamber as well asan epifluorescence staining technique, using the live/dead BacLightBacterial Viability Kit, has been studied in their work. The advan-tages of this method reported as follows: (i) it is a reliable, rapid,easy and yields both viable and total bacteria in only one step, (ii)samples are easy to prepare and easy to differentiate because of thehigh degree of contrast between the green color of the viablebacteria and the red color of the dead cells, (iii) BackLight stain doesnot produce background fluorescence.

Cultivation andmaintenance of AAB in pure culture is one of thegreatest hurdles, especially for strains isolated from high acetic acidlevel source (Sievers, Sellmer, & Teuber, 1992). The use of 20% maltextract as cryo protectant was effective for the preservation of allAAB strains (Sokollek et al.,1998a). Ndoye,Weekers, Diawara, Guiro,and Thonart (2007) used 20% mannitol as cryoprotectant to detectthe survival and preservation properties of thermoresistant AABafter freezeedrying process and found that freeze-dried cells couldbe conserved at 4 �C for at least 6 months without loss of viability.

Few ecological studies have analyzed the main AAB speciesinvolved in the process, while all studies have been conducted withcultivable strains only. The availability of a reliable and fast tech-nique for AAB enumeration is very useful in the food industry, inwhich AAB are used as biotechnological agents or in which AABmay spoil food product (Torija, Mateo, Guillamon, & Mas, 2010).

3.1. Molecular techniques

Classical microbiological taxonomy has traditionally usedmorphological, physiological and biochemical differences among thespecies to discriminate between them while physiological methods

Table 4Name of molecular techniques.

Name of molecular technique Reference

PCR-RFLP of the 16S rDNA/PCR-RFLP of the 16Se23S rDNA InternallyTranscribed Spacer (ITS)

Gonzalez, Guillamon, et aTrcek and Teuber (2002);

DNAeDNA Hybridizations Cleenwerck et al. (2002),Denaturing Gradient Gel Electrophoresis (DGGE)/

Temporal Temperature Gradient GelElectrophoresis (TTGE)

Andorra, Landi, Mas, GuillDe Vero and Giudici (2008

Fluorescence In Situ Hybridization (FISH) Blasco, Ferrer, and PardoReal-Time PCR (RT-PCR) Andorra et al. (2008), GamNested PCR Gonzalez, Hierro, et al. (2Epifluorescence Filter Technique Du Toit et al. (2005), MesRT-PCR with TaqManeMGB probe Torija et al. (2010)Amplified Fragment Length Polymorphism (AFLP)

DNA FingerprintingCleenwerck et al. (2009)

Random Amplified Polymorphic DNA-PCR(RAPD-PCR)

Bartowsky et al. (2003), N

Enterobacterial Repetitive IntergenicConsensus-PCR (ERIC-PCR)

Gonzales (2005), Nanda e

Repetitive Extragenic Palindromic-PCR (REP-PCR) Gonzales (2005)

would not be able to distinguish the currently described species(Gonzales, 2005). On the other hand, quantification of AAB in solidmedia has some limitations such as time requirement, inability todetect VBNC cells. Miniaturized phenotypic systems which wereinitially developed for the groups of bacteria (e.g. Enterobacteriaceae),such as ID32C, API 20 NE (bioMerieux) and RapID NH (Remel) werealso used for the identification of AAB without much success(Cleenwerck & de Vos, 2008). Finally, new techniques have beendevelopedusingmolecularapproaches toovercomethedisadvantagesof culturing and identification of AAB (Gonzalez, Hierro, Poblet,Mas, &Guillamon, 2006). Recently, the principles of currently applied meth-odology for characterization of AAB are summarized by Cleenwerckand de Vos (2008). These techniques are summarized in Table 4.

DNAeDNA hybridization values play a key role in species delin-eation. Several methods have been used to measure the DNA relat-edness among bacteria such as microplate method, the membranemethod, spectrophotometer initial renaturationmethod (Cleenwerck&deVos,2008). Lisdiyanti et al. (2001)delineatedAcetobacter species,isolated from Indonesian sources by DNAeDNA hybridization dataand Cleenwerck, Vandemeulebroecke, Janssens, and Swings (2002)confirmed the findings of Lisdiyanti et al. (2001). On the other hand,recently, a discrepancy, resulting in different taxonomic conclusionswas found between the DNA relatedness values reported by Dellaglioet al. (2005) and Lisdiyanti, Navarro, Uchimura, andKomagata (2006)with strains of Gluconacetobacter.

PCR-RFLP of the 16S rDNA and 16Se23S rDNA combining thedifferent restriction endonucleases, allow the identification of theAAB in a shorter period of time, compared with the time-consuming techniques (such as DNAeDNA hybridization). The useof these techniques for AAB differentiation is proposed for routinelaboratory analysis because of its easiness, the general use ofa single PCR and limited restriction analysis, and for the low cost ascompared to the techniques used to identify the novel AAB species(Gonzalez, Guillamon, et al., 2006; Trcek, 2005). Ruiz, Poblet, Mas,and Guillamon (2000) used this method for the identification ofAAB isolated from wine fermentations. In the study, the samedegree of distinction as that for the 16S rDNA analysis was obtainedwithin reference strains of AAB by PCR-RFLP of the 16Se23S rDNAITS. However, 16Se23S rDNA ITS restriction patterns of strainsisolated from wine did not mach those of any of the referencestrains. Thus, it is concluded that PCR-RFLP of the 16Se23S rDNAITS is not a useful method to identify isolates of AAB at the specieslevel, although it may be an adequatemethod to detect intraspecificdifferentiation (Ruiz et al., 2000).

l. (2006), Ruiz et al. (2000), Sievers, Lorenzo, Gianotti, Boesch, and Teuber (1996),Trcek (2005)

Lisdiyanti et al. (2001)amó n, and Esteve-Zarzoso (2008), De Vero et al. (2006);), Haruta et al. (2006), Ilabaca et al., 2008; Lopez et al. (2003)

(2003), Franke et al. (1999), Franke-Whittle, O’Shea, Leonard, and Sly (2005)mon et al. (2006), Gonzalez, Hierro, et al. (2006)

006)a, Macias, Cantero, and Barja (2003)

anda et al. (2001)

t al. (2001), Vegas et al. (2010)


AAB presence in Chilean vinegars was analyzed by using 16SrRNA gene, obtained by PCR amplifications of DNA and bacterialcompositionwas analyzed by RFLP-PCR of 16S rRNA gene, TemporalTemperature Gradient Gel Electrophoresis (TTGE) separation ofamplicons containing region V3eV5 of 16S rRNA gene and cloningof those amplicons (Ilabaca, Navarrete, Mardones, Romero, & Mas,2008). The result of the study showed that TTGE allows a quickinsight into overall and rough diversity, while cloning allowsenumeration of different species and observation of fine moleculardiversity. Thus, both methods complement each other in offeringa view of quantitative microbial diversity. Gluconacetobacter xyli-nus, Gluconacetobacter europaeus and Gluconacetobacter inter-medius could not be differentiated with the methods used, becauseof the sequence similarity of these species.

PCR-DGGE technique, which is useful to test the diversity of thebacterial community as TTGE, was used to determine the micro-bial population of rice vinegar and traditional balsamic vinegar(De Vero et al., 2006; Haruta et al., 2006). De Vero and Giudici(2008) reported that species, which are recovered from vinegarfermentation and mainly distributed in the genera Acetobacter,Gluconobacter and Gluconacetobacter, can be grouped morefrequently by PCR-DGGE. On the other hand, the limitation ofDGGE methods is that the identified species cannot be quantified.Only few studies developed methods for AAB quantification. Mostof the quantification methods such as Fluorescence in situhybridization (FISH), Epifluorescence and real-time PCR (RT-PCR)have been designed to detect the presence and quantify AAB asa group, without identification at the species level. However,recently, Torija et al. (2010) developed a rapid and specific quan-titative PCR to identify and quantify some AAB species present inwine and vinegar matrices. To do so, they designed TaqManeMGBprobes as highly sensitive and specific tool for A. pasteurianus,Acetobacter aceti, Gluconacetobacter hansenii, Ga. europaeus andGluconobacter oxydans, which are the main species detected inwine and vinegar, from the 16 S rRNA gene. The reliability of theprimers and probes designed to detect different species of AABwere confirmed by using previously identified 40 AAB strains withRFLPsePCR of 16S rRNA gene and 16S rRNA gene sequencing(Table 5).

RAPD-PCR (randomly amplified polymorphic DNA) has success-fully been used to differentiate A. pasteurianus strains isolated fromspoiled red wine (Bartowsky, Xia, Gibson, Fleet, & Henschke, 2003)and to characterize AAB in rice vinegar (Nanda et al., 2001). Ampli-fication of repetitive bacterial DNA elements through the poly-merase chain reaction (rep-PCR fingerprinting) using the (GTG)5primer ((GTG)5-PCR fingerprinting), was found a promising geno-typic tool for rapid and reliable speciation of AAB (De Vuyst et al.,2008). On the other hand, amplified fragment length poly-morphism (AFLP) DNA fingerprinting is found suitable for accurateidentification and classificationof a broad rangeofAAB, aswell as for

Table 5Species identification of AAB strains isolated during vinegar acetification by different tec

Origin of the isolates Number of isolates R

Wine vinegar (France) 19 ATraditional balsamic

vinegar (Italy)2 A

Wine vinegar (France) 4 GTraditional balsamic

vinegar (Italy)7 G

Submerged vinegar(Spain)

6 G

Wine vinegar (Spain) 1 GWine vinegar (Spain) 1 G

a No detection by any of five MGB probes tested.Table is taken from Torija et al. (2010

the determination of intraspecific genetic diversity (Cleenwerck, deWachter, Gonzalez, de Vuyst, & de Vos, 2009).

Real-time PCR and nested PCR for enumerating and detectingAAB were studied and no significant differences were foundbetween real-time PCR and the traditional techniques (colony andmicroscope counting). Nested PCR is also found useful for detectingAAB in environmental samples (Gonzalez, Hierro, et al., 2006).Although real-time PCR is accurate, it is also an expensive andcomplex technique. Restriction Fragment Length Polymorphism(RFLP) of amplified 16S rDNA, and amplification by polymerasechain reaction of Enterobacterial Repetitive Intergenic Consensus(ERIC-PCR), Repetitive Extragenic Palindromic (REP-PCR) and(GTG)5-rep-PCR techniques were successfully used to identify AABin wine and wine vinegar (Gonzalez, Hierro, Poblet, Mas, &Guillamon, 2005; Vegas et al., 2010). Both ERIC-PCR and (GTG)5-rep-PCR were used for typing AAB at the strain level and the resultsof both techniques were found similar, although ERIC-PCR yieldedslightly higher polymorphism (Vegas et al., 2010).

It can be seen that there are many methods to identify AABspecies. These group of bacteria strongly correlated at the phylo-genetic level and have phenotypic characteristics that are similar toone another. Therefore, a polyphasic study is the recommendedtechnique for an accurate identification of AAB strains (Cleenwerck& de Vos, 2008).

4. Sources of AAB

AAB are widespread in nature and a large number of strains ofAAB have been isolated fromvariety of sources (Table 6). As it can beseen from the table, AAB belonging to the genus Acetobacter havebeen isolated more frequently than those of Gluconobacter whileoccurrence of Acidomonas, Kozakia, Swaminathania, Saccharibacter,Neoasaia, Granulibacter, Tanticharoenia and Ameyamaea strains israther rare in common isolation sources. For example, the isolationsources of Acidomonas methanolica are quite unique. It could not beisolated from flowers and fruits. Acidomonas strains isolated mostlyfrom sludge (Uhlig, Karbaum, & Steudel, 1986; Urakami, Tamaoka,Suzuki, & Komagata, 1989). Yamada et al. (1999) were isolatedsixty-four of AAB from Indonesian sources such as fruits,flowers andfermented foods and identified them as Acetobacter strains (forty-five isolates), Gluconacetobacter strains (eight isolates) and Gluco-nobacter strains (eleven isolates). AABwere also isolated from fruitscollected in Thailand. Isolates, belong to A. pasteurianuswere foundin fruits of apple, banana, grape, guava, jack fruit, jujube, kaffir lime,langsat, longkong, longan, mango, mangosteen, orange, papaya,peach, pineapple, passion fruit, rose apple, rambutan, rakam, sapo-dilla, star gooseberry, strawberry, sugar apple, tamarind, watermelon, tomato and palm juice, while Acetobacter orientalis andGluconacetobacter liquefaciens were found in star fruits and palmjuice, respectively (Seearunruangchai et al., 2004).

hniques.

FLPsePCR 16S rRNA gene RT-PCR with TaqManeMGB probe

. pasteurianus A. pasteurianus

. pasteurianus A. pasteurianus

a. europaeus Ga. europaeusa. europaeus Ga. europaeus

a. europaeus Ga. europaeus

. oxydans G. oxydansa. intermedius Nda

).

Table 6Isolation sources of acetic acid bacteria.

Sources Species Reference

Coffea arabica Ga. diazotrophicus Jimenez-Salgado et al. (1997),Madhaiyana et al. (2004)

Coffee plants Ga. azotocaptans,Ga. johannae

Fuentes-Ramírez et al., 2001

Fermented cocoabean

A. pasteurianus,A. syzgii orA. lovaniensis-like andA. tropicalis-like;A. syzygii,A. pasteurianus,A. tropicalis,A. malorum,G. oxydansA. aceti,A. pasteurianusA. lovaniensis

Ardhana and Fleet (2003),Carr et al. (1979),De Vuyst et al. (2008),Lagunes-Gálvez et al. (2007),Nielsen et al. (2007), Schwanand Wheals (2004)

Tropical fruits(coconut, mango,guava, sapodillaetc.)

A. orleanensis,A. lovaniensis,A. syzygii,A. indonesiensis,A. cibinongensis,A. tropicalis,A. orientalis,G. oxydans,G. frateurii,Ga. hansenii,F. aurantia

Lisdiyanti et al. (2003)

Rotting apple A. malorum Cleenwerck et al. (2002)Corn roots G. azotocaptans Menhaz, Weselowski, and

Lazarovits (2006)Cherry (Prunus sp.) G cerinus Yamada and Akita (1984)Strawberry G. frateurii Mason and Claus (1989)Grape G. oxydans,

A. acetiJoyeux et al. (1984),Gonzales (2005)

Dried fruit Ga. liquefaciens Yamada et al. (1997)Flowers (ixora,

lantana etc.)A. orleanensis,A. indonesiensis,A. syzygii,A. orientalis,G. oxydans,G. frateurii,As. bogorensis,As. siamensis,As. indonesiensis,F. aurantia

Lisdiyanti et al. (2003),Yukphan et al. (2009)

Flowers of redginger

Am. chiangmaiensis

Pollen Sa. floricola Jojima et al. (2004)Beer A. cerevisiae

A. pasteurianus,G. oxydans

Cleenwerck et al. (2002),Skerman, McGowan, andSneath (1980)

Sugarcane roots Ga. diazotrophicus Yamada et al. (1997)Mealy bug from

sugar caneGa. sacchari Franke et al. (1999)

Palm brown sugar,ragi

K. baliensis Lisdiyanti et al. (2003)

Wetland rice G. diazotrophicus,A. peroxydans

Muthukumarasamyet al. (2005)

Wild rice Sw. salitolerans Loganathan andNair (2004)

Abbreviations: A.: Acetobacter, G.: Gluconobacter, Ga.: Gluconacetobacter,K.: Kozakia, As.: Asaia, Am.: Ameyamaea, Sa.: Saccharibacter, Sw.: Swaminathania,F.: Frateuria (this genus differently classified into the Gammaproteobacteria).


5. Beneficial effects of AAB in foods

Although AAB can be isolated from various natural sources,they are frequently isolated in alcoholic juices such as hard cideror wine, or from beer. AAB can play not only a positive role in theproduction of selected foods and beverages, but they can alsospoil other foods and beverages, such as wine, beer, soft drinks,and fruits. AAB of the genera Acetobacter and Gluconobacter are

known as a spoiling agent of beer and wine. Gluconobacter spp.,which are resistant to preservatives such as sorbic acid, benzoicacid, and dimethyldicarbonate, are the most frequently encoun-tered cause of bacterial spoilage of soft drinks at low pH (Raspor& Goranovic, 2008). When basic hygienic and technical proce-dures are not correctly performed, AAB produce acetic acid inbeverages under aerobic/microaerophilic conditions and causelow pH and ethanol content, pack swelling, vinegary off-flavors,turbidity and ropiness (Guizani & Mothershaw, 2006; Odhav,2004; Stratford & Capell, 2003).

Cultures of AAB are used in the commercial production ofvinegar. The oxidation of ethanol to acetic acid, which is importantin vinegar production, is the best known characteristics of AAB.However, these bacteria also oxidize glucose to gluconic acid,galactose to galactonic acid, arabinose to arabonic acid, and so on.This property of “underoxidation” is exploited in the manufactureof ascorbic acid (vitamin C). Ascorbic acid can be formed fromsorbose, but chemical synthesis of sorbose is difficult. It is, however,conveniently obtainable from AAB, which oxidize sorbitol (a readilyavailable sugar alcohol) only to sorbose, a process called biocon-version. Another interesting property of some AAB is their ability tosynthesize cellulose. The formed cellulose does not differ signifi-cantly from that of plant cellulose, with the exception that it is pureand not mixed with other polymers and is formed as a matrixoutside the wall where the bacteria become embedded in thespecies of AAB grow in an unshaken vessel. They form a surfacepellicle of cellulose in which the bacteria develop (Madigan &Martinko, 2006).

Researchers that focused on the isolation and identification ofAAB, have mainly investigated vinegar, wine, cocoa and coffee asa research material because of the industrial values they have.These studies are summarized below:

5.1. AAB in vinegar

The best known industrial application of AAB is vinegarproduction. Several types of vinegars are produced worldwide;they differ for raw materials, technologies and use (Solieri &Giudici, 2009). There are two well defined methods to producevinegar: the traditional and the submerged. In the submergedmethod, which has a short production time (24e48 h), AAB aresubmerged in the liquid and oxygen is constantly added constantly.In traditional method also called as “surface culture method” AABgrow on the media surface where oxygen concentration is high.Therefore, acetic acid production takes long time and resultingvinegar is of high quality (Tesfaye, Morales, Garcίa-Parrilla, &Troncoso, 2002). In traditional methods, oxidation is started by“seed-vinegar”, also called “mother of vinegar”, an undefinedstarter culture obtained from previous vinegar.

Few ecological studies of AAB in vinegars have been made, andthoseweremainly focused on industrial vinegars, persimmonvinegar,rice vinegar, wine vinegar and traditional balsamic vinegar (Vegaset al., 2010). In the early studies, AAB isolated from vinegar fermenta-tions were identified as Acetobacter acidophilum, (Wiame, Harpigny, &Dothey, 1959), Acetobacter polyoxogenes (Entani, Ohmori, Masai, &Suzuki, 1985), A. hansenii and A. pasteurianus (Kittelmann, Stamm,Follmann, & Triiper, 1989). However, studies focused on identifica-tion of AAB are spread on awide period of timewith a significant gapbetween older papers and current AAB taxonomy (Table 7).

The population dynamics of AAB in traditional wine vinegar,which is obtained by spontaneous wine acetification, was studiedby Vegas et al. (2010). In this study, themain species throughout theprocess was found as A. pasteurianus which has been mentioned ina previous study in traditional wine vinegar (Ilabaca et al., 2008)and rice vinegar production (Haruta et al., 2006; Nanda et al., 2001).

Table 7AAB species isolated from different kinds of vinegar.

Source Species Reference

Rice vinegar A. pasteurianus Haruta et al. (2006), Nanda et al. (2001)Industrial vinegar Ga. europaeus

A. oboediens, A. pomorumA. intermediusGa. entaniiGa. europaeus, Ga. hansenii

Boesch, Trcek, Sievers, and Teuber (1998), Schüller, Hertel, and Hammes (2000),Sievers et al. (1992), Sokollek et al. (1998b), Yamada et al., 1997

Traditional balsamic vinegar Ga. xylinus, A. acet, A. pasteurianusGa. europaeus, Ga. hanseniiA. pasteurianu, A. malorum

Gullo et al. (2006), Gullo and Giudici (2006)

Traditionalwine vinegar

A. pasteurianus, Ga. europaeus Ilabaca et al. (2008), Vegas et al. (2010)

Abbreviations: A.: Acetobacter; Ga.: Gluconacetobacter.


Vegas et al. (2010) observed that Ga. europaeus was proliferatedwhen the acetic acid concentration was appropriate, in their study.Thus, it is concluded that the starting strains were better preparedto survive at low acetic acid concentrations and high ethanol,whereas the ones that finished the acetification might be moreacetic acid tolerant. They also reported that the longer operating ofwine vinegar may lead to a greater diversity of AAB strains, whichimproves the chances of a good acetification.

Using AAB as a starter culture in the production of vinegar maylead to an improved fermentation process and an enhancedproduct quality (Gullo & Giudici, 2008). Zahoor, Siddique, andFarooq (2006) reported that, in Pakistan, a mixed culture ofAcetobacter is used for the production of acetic acid but no attentionis given towards its proper maintenance and culture is contami-nated with other kinds of microorganisms. So for that reason, theyconducted a research to isolate a pure culture of A. aceti for vinegarproduction and produced vinegar from the pure culture obtained,under controlled conditions, which has better organoleptic prop-erties than commercial vinegar.

Although vinegar is used for giving taste to foods, it is also usedfor sanitizing effect, especially for salad vegetables, which areconsumed raw (Sengun & Karapinar, 2004, 2005a, 2005b). It isreported that AAB, that produce acetic acid during the fermentationperiod, are mainly responsible from the sanitizing effect of vinegar(Costa, Thomaz-Soccol, Paulino, & de Costa, 2009; Karapinar &Gonul, 1992a, 1992b).

5.2. AAB in cocoa

Characteristic flavor of cocoa is developed through a fermenta-tion of cocoa beans. At the beginning of fermentation yeast and LABare dominated because of the low pH value and the high sugarcontent of the pulp while AAB predominate with temperature risesand alcohol accumulates (Biehl & Ziegleder, 2003; Nielsen et al.,2007; Thompson, Miller, & Lopez, 2001).

Growth behaviour of AAB during different stages of cocoafermentationwas studied bymany researchers. Nielsen et al. (2007)reported that Acetobacter syzygii, A. pasteurianus and A. tropicaliswere the predominant AAB during Ghanaian cocoa fermentation.Similar fermentationwas investigated by De Vuyst et al. (2008) andA. pasteurianus, A. syzgii or A. lovaniensis-like and A. tropicalis-likestrains were identified from Ghanaian cocoa bean fermentation.A. pasteurianus and A. aceti strains have been reported to forma significant part of the AAB community during cocoa fermentationsin Ghana (heap), Indonesia (box) and Brazil (box) (Ardhana & Fleet,2003; Carr, Davies, & Dougan, 1979; Schwan & Wheals, 2004).A. lovaniensis was isolated from the cocoa fermentation in theDominican Republic as the predominating AAB (Lagunes-Gálvez,Loiseau, Paredes, Barel, & Guiraud, 2007). Although Acetobecterspp. were mainly isolated from cocoa productions as predominant

AAB, Gluconobacter spp. were also found in cocoa fermentations(Biehl & Ziegleder, 2003; Nielsen et al., 2007).

5.3. AAB in coffee

Coffea arabica (arabica) and C. canephora (robusta), which aredominant Coffea species, are processed by wet or dry methods(Schwan & Wheals, 2003). The microbiota involved in ‘dry’ pro-cessing aremuchmore varied and complex than those found duringwet fermentation, but the role of microorganisms found in coffeefermentation by natural processing is still unknown (Silva, Batista,Abreu, Dias, & Schwan, 2008). Microbial profile of AAB in coffeefermentation was studied by Silva et al. (2008), Silva, Schwan, Dias,andWheals (2000). In these studies, bacteria, yeast and filamentousfungi were isolated during coffee processing, while no AAB wereidentified. On the other hand, two species within the genus Gluco-nacetobacter, associated to coffee plants, were described in Mexico:Ga. johannae andGa. azotocaptans (Fuentes-Ramírez et al., 2001).Ga.diazotrophicus, which is known as nitrojen-fixing AAB, were alsoisolated from coffee plants (C. arabica L.) (Jimenez-Salgado et al.,1997; Madhaiyana et al., 2004).

5.4. AAB in wine

AAB play a negative role in winemaking since they increase thevolatile acidity of wines. They can survive in the various phases ofalcoholic fermentation and it is very important to control theirpresence and ulterior development to obtain good quality wines(Gonzalez, Hierro, et al., 2006).

Du Toit and Lambrechts (2002) investigated the occurrence ofAAB in South African red wine fermentations. The initial AABnumbers were found ranging from 106e107 (for 1998 must) to104e105 cfu/ml (for the 1999 must), decreasing 102e103 cfu/ml inmusts having a low pH (�3.6) and increasing during fermentation.Many researchers have identified different strains of AAB, found inwine fermentations (Table 8).

Growth behaviour of AAB during different stages of vinificationwas studied by Gonzalez et al. (2005). G. oxydans was foundpredominant species in fresh must and the first stages of fermen-tation. On the other hand, only few species survived the transferfrom grapes to must and the ones that did were mostly A. acetispecies. It is reported that the anaerobic conditions of the alcoholicfermentation make conditions unsuitable for the growth of AAB.When the wine is transferred from fermentation tanks to otherstorage vessels, agitation and aeration processes encourage thegrowth of surviving AAB populations (Drysdale & Fleet, 1988).Bacterial spoilage has also recently been reported to occur in pack-agedwine such as vertically upright bottles (Bartowsky et al., 2003).This is visually evident as a distinctive ring of bacterial biomass thatis deposited on the neck of the bottle at the interface between the

Table 8AAB species isolated from different kinds of wine.

Source Species Reference

Bottled red wine A. pasteurianus Bartowsky et al. (2003)Red wine fermentation G. oxydans, Ga. hansenii, A. aceti Gonzales (2005)

A. nitrogenifigens Dutta and Gachhui (2006)Dao region of Portugal red wine A. oeni Silva et al. (2006)South African red wine G.oxydans, A. pasteurianus, A. hansenii,

A. liquefaciensDu Toit and Lambrechts (2002)

Austrian wine A. tropicalis Silhavy and Mandl (2006)White wine G.oxydans, A. pasteurianus, A. aceti Joyeux et al. (1984)


wine and the air headspace.Higher temperature ofwine storage andhigher wine pH favored the development and the metabolism ofAAB (Joyeux, Lafon-Lafourcade, & Ribereau-Gayon, 1984).

The factors affecting the development of AAB during the wine-making process are the pH of the must and wine, the temperature,the ethanol concentration and the dissolved oxygen in the media(Drysdale & Fleet,1988). In redwines, AABwere studied during agingin barrels and enumeration on plate counts showed that the level ofpopulationwas closely linked to the dissolution of oxygen. Howeverit is also found that they were always present even in the absence ofoxygen in very low levels, whichmeans that a large proportion of thepopulationwas in VBNCwhen deprived of oxygen (Funel, 2005). It isreported that the presence of these strictly aerobic bacteria in grapemust and during wine maturation can be controlled by eliminating,or at least limiting oxygen, as essential growth factor. The effects ofSO2, which is used as an antioxidant and antimicrobial agent duringwinemaking, on the survival and growth of AAB have not been wellinvestigated.A recent studydemonstrated that lowconcentrations ofSO2 (0.35 mg/l molecular SO2) were found to have minimal effect onviability and cultivability of an A. pasteurianus strain, whereas higherconcentrations were effective (1.2 mg/l molecular SO2) (Du Toit,Pretorius, & Lonvaud-Funel, 2005). Ubeda and Briones (1999)screened 68 samples of filtered and unfiltered wines for the pres-ence of AAB and observed that 13% of the unfilteredwines containedAAB while filtered samples were not containing them. Althoughexclusion by sterile filtration is a possible control measure,membrane fouling, cost and time delays can limit application withredwines. Blanketingwinewith an inert gas such as carbon dioxide,and ensuring that storage containers are completely filledwithwineto minimize contact the headspace of air can be applied to preventthe growth of AAB (Bartowsky & Henschke, 2008).

6. Conclusion

The classification of AAB has not been fully established yet. Thetaxonomic resolution of molecular techniques used for the char-acterization of AAB is not clear. Polyphasic analysis is recom-mended as the best approach for classifying this group of bacteria.Researchers mainly focused on wine and vinegar associated AABwhich have a great importance in food industry. They are respon-sible for vinegar fermentation producing acetic acid from ethanolwhile they are found in wine in a detrimental way. Isolation,cultivation and preservation difficulties restrict the usage of AAB invinegar production as a starter culture. Future studies shouldovercome these disadvantages and focus on the starter cultureselection and process set up for better process control in vinegarproduction.

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Acetic Acid Bacteria